Aggrewell well plates

Patient-derived GBM cell lines, HK217 (proneural), HK301 (proneural) and HK280 (mesenchymal) were used for encapsulation. Sizes of GBM spheroids were standardized by seeding approximately 600K cells per well into Aggrewell™ well plates (Stemcell Technologies) one day prior to encapsulation. The following day, spheroids were harvested from the wells, centrifuged briefly (200×G, 1 min) and resuspended in the hydrogel precursor solution. Spheroid-laden hydrogels were formed as described in the above hydrogel fabrication section. Cell migration was observed periodically (Imaged at Day 1,3,6 and 9) by acquiring phase contrast images on a Zeiss Axio.Z1 Observer microscope with a Hamamatsu Orca Flash 4.0 V2 Digital CMOS Camera and Zeiss ZEN 2 (Blue Edition) software. Quantitation is described separately. At the end of experimental period, hydrogels were fixed using 4% paraformaldehyde (PFA) and stained with Hoescht (nuclei) and Cell Mask™ (cell membrane). These gels were imaged using a Leica SP5 confocal microscope. To block ITGαV-IBSP interaction, GBM spheroids were incubated with 10 μg/mL anti-ITGαV antibody a day prior to encapsulation. In addition, after the encapsulation, hydrogels were incubated in GBM media containing 10 μg/mL of the same antibody over the course of the experiment.